Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Abstract

Summary Bread wheat ( Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression ( CE ) of the rice ( Oryza sativa L.) nicotianamine synthase 2 ( Os NAS 2 ) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine ( NA ) and 2′‐deoxymugineic acid ( DMA ) – that play key roles in metal transport and nutrition. The CE ‐ Os NAS 2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy ( XFM ) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE ‐ Os NAS 2 grain and positively correlated with NA and DMA concentrations.

Nicotianamine-chelated iron positively affects iron status, intestinal morphology and microbial populations in vivo (Gallus gallus)

“…Zinc deficiency in chickens is known to negatively alter the gut microbiome, and improved microbial composition in 'Biofortified' chickens may be an additional symptom of improved Zn status 75 . Given NA and DMA enhance Fe bioavailability we have previously argued these natural metal chelators function as phytonutrients in cereal foods 25,26,76 . It is well established that NA exhibits anti-hypertensive effects in vivo 34,77 although we did not detect differences in heart angiotensin-converting enzyme (ACE) and angiotensin II receptor type 1 (AT1R) gene expression throughout our study (Figs.…”

“…Plant material and white flour production. Vector construction, plant transformation and the initial selection of biofortified wheat material is described in 25 . In brief, the full-length coding sequence of OsNAS2 (LOC_Os03g19420) was PCR amplified from rice (Orzya sativa L.) cv.…”

“…Total dietary fiber was measured via enzymatic digestion using heat-resistant amylase, protease and amyloglucosidase according to manufacturer's instructions (Total Dietary Fiber Assay Kit, Sigma, St. Louis, MO, USA). Quantification of NA and DMA in diet and flour samples was performed as described in 25 . Briefly, sequential MeOH (100%) and 18MΩ H 2 O sample were derivatized by 9-fluorenylmethoxycarboxyl chloride (FMOC-Cl) and quantified via RP LC-MS on a 1290 Infinity II and 6490 Triple Quadrupole LC/MS system (Agilent Technologies Inc., Santa Clara, CA, USA).…”

“…Caco-2 Fe bioavailability bioassay. Diet and flour samples were subjected to the Caco-2 cell bioassay as previously described 25 . Briefly, gastric-digested samples (1.5 mL) were added to cylindrical Transwell inserts (Corning Life Sciences, Corning, NY) fitted with a semipermeable (15 000 Da MWCO) basal membrane (Spectra/ Por 2.1, Spectrum Medical, Gardena, CA) and placed within wells containing Caco-2 cell monolayers.…”

“…In graminaceous cereals NA serves as the biosynthetic precursor to 2′-deoxymugenic acid (DMA), a related Fe chelator in plant tissues that also functions as a root-secreted phytosiderophore to chelate ferric Fe in the rhizosphere 23 . Both NA and/or DMA are major Fe chelators in white wheat (Triticum aestivum L.) flour and enhancers of in vitro Fe bioavailability [24][25][26] and increased NA/DMA biosynthesis has been employed to biofortify wheat 25,27 and rice (Oryza sativa L.) [28][29][30] with Fe and Zn. While both NA and DMA chelate ferric (Fe 3+ ) ions, only NA is capable of chelating highly-bioavailable Fe 2+ ions 31,32 .…”